We will explore and determine the molecular mechanism for epitope shift, a recently recognized feature of affinity maturation in which somatic hypermutation changes the epitope recognized by mature antibodies from that recognized by germline antibodies. Our published work indicates that affinity maturation leads to both affinity and epitope selection changes, both of which can be critical for developing neutralizing antibodies. To date, this has been unappreciated because previous studies of affinity maturation have focused on cases where maturation improves affinity to an essentially identical epitope. This is the case both for protein antigens and haptens, as exemplified by structural and biochemical studies using hen egg white lysozyme and small haptens. In these cases, affinity maturation improves binding to identical epitopes. The anti-lysozyme antibodies are particularly interesting in this light, because they use large (~1,800E2) interfaces with essentially identical interactions where enhanced affinity arises from increased burial of hydrophobic surface at the edge of the interaction surface. These changes are driven by subtle rearrangements of non-hotspot residues. While the edge of the interaction surface changes in the anti- lysozyme antibodies, these changes do not lead to changes in the epitope recognized. In our model, this is because hot-spot residues are germline encoded. Our proposed work will explore changes hypothesized to occur within the antibody combining site and establish the mechanism whereby these changes cause epitope shifting. We will focus anti-rotavirus VP6 antibodies that use the VH1-46 gene segment. VP6 is the immunodominant epitope and VH1-46 is the dominant heavy chain gene segment seen in the antibody response against rotavirus. In general, adults produce neutralizing antibodies to rotavirus, but infants do not. This is not due to germline gene usage, but rather it is to more somatic hypermutation in adults. Rotaviruses are the most important cause of severe diarrhea in infants and young children in both the developed world and the developing world. Attenuated virus vaccines have been introduced, however even when properly administered in a controlled setting, these vaccines appear to reduce infection by only ~61%. Additionally, they offer no protection against emerging strains, and require multiple (2 or 3) doses. Second generation vaccines with improved efficacy are sought, and subunit vaccines are an attractive choice. CDC estimates are that even with vaccination ~500,000 people still die annually from rotavirus infection. Effective development of subunit vaccines is aided by a comprehensive understanding of the mechanisms the human immune system employs to recognize antigens and neutralize pathogens. This application will provide molecular immunology paradigms that may influence the choice of antigens, the use of surrogate markers of protection, and the development of new rotavirus vaccines. We will explore and determine the molecular basis for epitope shift. PUBLIC HEALTH RELEVANCE: Despite recent vaccines, rotaviruses remain the largest cause of severe diarrhea, with estimates indicating that vaccines are likely 45-61% effective, the CDC estimates that rotavirus still claims >500,000 lives annually, mostly in children younger than 5 years in the developing world. The antibody immune response to this disease is impaired in infants, such that infants use the same dominant germline genes, but have impaired affinity maturation processes and do not produce neutralizing antibodies. We will determine the mechanism of a newly identified aspect of affinity maturation, epitope shift, and provide new molecular immunology paradigms that may influence the choice of antigens, the use of surrogate markers of protection, and the development of new rotavirus vaccines.